High Dynamic Range Digital Assay Enabled by Dual-Volume Centrifugal Step Emulsification.

Area of Science:

• Microfluidics
• Biotechnology
• Assay Development

Background:

• Digital assays require a wide dynamic range for accurate quantification.
• Existing multi-volume approaches often involve complex manufacturing or specialized equipment.

Purpose of the Study:

• To develop a novel dual-volume centrifugal step emulsification technique on a single chip.
• To extend the dynamic range of digital assays without increasing manufacturing complexity.

Main Methods:

• Implementing dual-volume centrifugal step emulsification on a microfluidic chip.
• Automated sample metering into two subvolumes, droplet generation, and monolayer alignment.
• Demonstrating digital quantification using droplet digital loop-mediated isothermal amplification (ddLAMP).

Main Results:

• Extended dynamic range by two orders of magnitude compared to single-volume methods.
• Generated 2,500 and 176,000 droplets from 10.5 μL and 2.5 μL subvolumes, respectively.
• Achieved quantification over 5 log steps with linearity (R² ≥ 0.992) after 30 min incubation.

Conclusions:

• Dual-volume centrifugal step emulsification offers a simplified, effective method for extending digital assay dynamic range.
• This technique overcomes limitations of existing multi-volume approaches, requiring neither complex manufacturing nor specialized equipment.
• The microfluidic chip design automates key steps, enabling efficient and precise droplet generation for digital quantification.